Revista Portuguesa de Endocrinologia, Diabetes e Metabolismo...1646-3439/© 2019 Sociedade...

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128 Rev Port Endocrinol Diabetes Metab. 2019;14(2) Revista Portuguesa de Endocrinologia, Diabetes e Metabolismo www.spedmjournal.com Artigo Original INFORMAÇÃO SOBRE O ARTIGO https://doi.org/10.26497/ao180019 1646-3439/© 2019 Sociedade Portuguesa de Endocrinologia, Diabetes e Metabolismo. Publicado por Sociedade Portuguesa de Endocrinologia, Diabetes e Metabolismo. Este é um artigo Open Access sob uma licença CC BY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/4.0/). * Autor Correspondente / Corresponding Author. E-Mail: [email protected] (Cláudio Daniel Cerdeira) Faculty of Biomedical Sciences, José do Rosário Vellano University (Unifenas/Alfenas), MG-179, Km 0, 37132-440, Alfenas, Minas Gerais, Brazil. Palavras-chave: Antioxidantes; Diabetes Mellitus Experimental; Ratos Wistar. Keywords: Antioxidants; Diabetes Mellitus Experimental; Rats Wistar. RESUMO ABSTRACT Introduction: Diabetes mellitus (DM) is still considered a major public health concern worldwide and lacks new and safe drugs for long-term and/or adjuvant therapy. Thus, we postulated that nitrox- ides could prevent DM complications in rats. Methods: To evaluate the effects of the nitroxides tempo and tempol in preventing hyperglycemia, heart damage, DM-associated oxidative stress-induced renal injury, and other DM complications, nitroxides (30 mg/kg/day) were fed to Wistar rats with alloxan-induced type 1 DM for 60 days. The animals were then evaluated for glucose levels, lipid peroxidation in the kidneys, biochemical pa- rameters of renal function (creatinine levels), liver function (aspartate aminotransferase [AST] and alanine aminotransferase [ALT] total lipids, and steatosis), and lipid profiling (total cholesterol and fractions), and a histological analysis of the kidneys, liver, heart, and eyes was performed. Results: Treatment of the diabetic rats with tempo or empol prevented hyperglycemia (p < 0.01). Interestingly, only tempo was able to prevent lipid peroxidation in the kidneys of the diabetic rats (p < 0.01), while also decreasing creatinine levels (p < 0.05). In contrast, tempol prevented damage in the liver, kidneys, eyes, and heart of diabetic rats (p < 0.01) and showed evidence of preventing left ventricular hypertrophy. Notably, tempol and tempo prevented any significant increase in ALT levels and in the amount of total lipids in the liver of the diabetic rats. Conclusion: tempo and tempol presented great potential for preventing hyperglycemia and its com- plications in rats, both of which decreased markers of liver dysfunction. Tempo also mitigated oxida- tive damage to the kidneys, while tempol considerably attenuated heart injury. Nitróxidos Melhoram os Parâmetros Bioquímicos e Previnem ILesões em Orgãos de Ratos com Diabetes Mellitus Tipo 1 Introdução: A diabetes mellitus (DM) ainda é considerada uma das principais preocupações de saú- de pública em todo o mundo e carece de medicamentos novos e seguros para uma terapia de longo prazo e/ou adjuvante. Assim, postulamos que os nitróxidos poderiam prevenir as complicações do DM em ratos. Métodos: Para avaliar os efeitos dos nitróxidos tempo e tempol na prevenção de hiperglicemia, lesão cardíaca, lesão renal induzida por estresse oxidativo e outras complicações do DM, os nitróxidos (30 mg/kg/dia) foram administrados durante 60 dias a ratos Wistar com DM tipo 1 induzido por Historial do artigo: Received/ Recebido: 2017-12-29 Accepted/Aceite 2019-04-22 Online: 2020-01-22 © Autor (es) (ou seu (s) empregador (es)) 2019. Reutilização permitida de acordo com CC BY-NC. Nenhuma reutilização comercial. © Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. Nitroxides Improve Biochemical Profiling and Prevent Injuries in the Organs of Type 1 Diabetic Rats Anelise R Brito a , Gabriela F Ravazi a , Ana PP de Oliveira a , Cláudio Daniel Cerdeira a * , Bruno CC Salles b , Eduardo P Camara a , Jyean Muchon a , Gabriel OI Moraes b , Fernanda BA Paula c , Maísa RPL Brigagão b , Gérsika B Santos a a Research Laboratory in Biological Sciences, José do Rosário Vellano University (Unifenas), Alfenas, Minas Gerais, Brazil. b Faculty of Biomedical Sciences, José do Rosário Vellano University (Unifenas), Alfenas, Minas Gerais, Brazil. 3Laboratory of Clinical Biochemistry, Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences, Federal University of Alfenas, Minas Gerais, Brazil.

Transcript of Revista Portuguesa de Endocrinologia, Diabetes e Metabolismo...1646-3439/© 2019 Sociedade...

Page 1: Revista Portuguesa de Endocrinologia, Diabetes e Metabolismo...1646-3439/© 2019 Sociedade Portuguesa de Endocrinologia, Diabetes e Metabolismo. Publicado por Sociedade Portuguesa

128 Rev Port Endocrinol Diabetes Metab. 2019;14(2)

Revista Portuguesade Endocrinologia, Diabetes e Metabolismo

www.spedmjournal.com

Artigo Original

INFORMAÇÃO SOBRE O ARTIGO

https://doi.org/10.26497/ao1800191646-3439/© 2019 Sociedade Portuguesa de Endocrinologia, Diabetes e Metabolismo. Publicado por Sociedade Portuguesa de Endocrinologia, Diabetes e Metabolismo. Este é um artigo Open Access sob uma licença CC BY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Autor Correspondente / Corresponding Author.E-Mail: [email protected] (Cláudio Daniel Cerdeira)Faculty of Biomedical Sciences, José do Rosário Vellano University (Unifenas/Alfenas), MG-179, Km 0, 37132-440, Alfenas, Minas Gerais, Brazil.

Palavras-chave:Antioxidantes; Diabetes Mellitus Experimental; Ratos Wistar.

Keywords:Antioxidants; Diabetes Mellitus Experimental;Rats Wistar.

R E S U M O

A B S T R A C T

Introduction: Diabetes mellitus (DM) is still considered a major public health concern worldwide and lacks new and safe drugs for long-term and/or adjuvant therapy. Thus, we postulated that nitrox-ides could prevent DM complications in rats. Methods: To evaluate the effects of the nitroxides tempo and tempol in preventing hyperglycemia, heart damage, DM-associated oxidative stress-induced renal injury, and other DM complications, nitroxides (30 mg/kg/day) were fed to Wistar rats with alloxan-induced type 1 DM for 60 days. The animals were then evaluated for glucose levels, lipid peroxidation in the kidneys, biochemical pa-rameters of renal function (creatinine levels), liver function (aspartate aminotransferase [AST] and alanine aminotransferase [ALT] total lipids, and steatosis), and lipid profi ling (total cholesterol and fractions), and a histological analysis of the kidneys, liver, heart, and eyes was performed. Results: Treatment of the diabetic rats with tempo or empol prevented hyperglycemia (p < 0.01). Interestingly, only tempo was able to prevent lipid peroxidation in the kidneys of the diabetic rats (p < 0.01), while also decreasing creatinine levels (p < 0.05). In contrast, tempol prevented damage in the liver, kidneys, eyes, and heart of diabetic rats (p < 0.01) and showed evidence of preventing left ventricular hypertrophy. Notably, tempol and tempo prevented any signifi cant increase in ALT levels and in the amount of total lipids in the liver of the diabetic rats. Conclusion: tempo and tempol presented great potential for preventing hyperglycemia and its com-plications in rats, both of which decreased markers of liver dysfunction. Tempo also mitigated oxida-tive damage to the kidneys, while tempol considerably attenuated heart injury.

Nitróxidos Melhoram os Parâmetros Bioquímicos e Previnem ILesões em Orgãos de Ratos com Diabetes Mellitus Tipo 1

Introdução: A diabetes mellitus (DM) ainda é considerada uma das principais preocupações de saú-de pública em todo o mundo e carece de medicamentos novos e seguros para uma terapia de longo prazo e/ou adjuvante. Assim, postulamos que os nitróxidos poderiam prevenir as complicações do DM em ratos. Métodos: Para avaliar os efeitos dos nitróxidos tempo e tempol na prevenção de hiperglicemia, lesão cardíaca, lesão renal induzida por estresse oxidativo e outras complicações do DM, os nitróxidos (30 mg/kg/dia) foram administrados durante 60 dias a ratos Wistar com DM tipo 1 induzido por

Historial do artigo:Received/ Recebido: 2017-12-29Accepted/Aceite 2019-04-22Online: 2020-01-22

© Autor (es) (ou seu (s) empregador (es)) 2019. Reutilização permitida de acordo com CC BY-NC. Nenhuma reutilização comercial.© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC.No commercial re-use.

Nitroxides Improve Biochemical Profi ling and Prevent Injuries in the Organs of Type 1 Diabetic Rats

Anelise R Britoa, Gabriela F Ravazia, Ana PP de Oliveiraa, Cláudio Daniel Cerdeiraa *, Bruno CC Sallesb, Eduardo P Camaraa, Jyean Muchona, Gabriel OI Moraesb, Fernanda BA Paulac, Maísa RPL Brigagãob, Gérsika B Santosa

a Research Laboratory in Biological Sciences, José do Rosário Vellano University (Unifenas), Alfenas, Minas Gerais, Brazil.b Faculty of Biomedical Sciences, José do Rosário Vellano University (Unifenas), Alfenas, Minas Gerais, Brazil.3Laboratory of Clinical Biochemistry, Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences, Federal University of Alfenas, Minas Gerais, Brazil.

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Introduction

Diabetes mellitus (DM) is a group of endocrine and metabol-ic diseases affecting carbohydrate metabolism and is defined by elevated glycemic markers. The genesis of DM can occur from impaired insulin secretion by the pancreas (type 1 DM or insulin-dependent DM), resistance to insulin action (type 2 DM or non-insulin-dependent DM), or both. Currently, DM is still considered a major public health concern worldwide and is one of the leading causes of death globally.1-3

Complications from DM are divided into microvascular (e.g., retinopathy, blindness, nephropathy, and neuropathy) and macro-vascular (heart attacks/injury, left ventricular hypertrophy [LVH], strokes, and peripheral vascular disease).4 The associated ne-phropathy is one of the most serious and common microvascular complications of DM, and it is considered the leading cause of chronic renal failure (CRF) as well as the main indication for dial-ysis and transplantation. Approximately 30% of patients with DM develop diabetic nephropathy. Throughout the world, this chronic complication of DM affects approximately 25% of individuals with type 1 DM and approximately 5% to 10% with type 2 DM.3

During the development of chronic diseases such as DM (in-cluding diabetic heart disease [DHD]), the increase in production of reactive oxygen/nitrogen species (ROS/RNS) is remarkable, and the consequent so-called “oxidative stress” and oxidative tis-sue damage are common.5,6 Indeed, for long-term complications of DM, oxidative stress, inflammation, and the formation of ad-vanced glycation end-products (AGEs) have been reported. Thus, hyperglycemia in uncontrolled DM is known to induce oxidative stress through glucose-induced ROS, which can cause oxidative damage in the kidneys and heart.5‒7 Recent studies have revealed the important contribution of renal oxidative stress in the patho-genesis of diabetic nephropathy and chronic kidney disease.8,9 Among ROS, the superoxide (O2‒•) is critically important in the regulation of renal function through diverse mechanisms related to the control of blood pressure.10

Nicotinamide adenine dinucleotide phosphate (NADPH) oxi-dase complex (i.e., Nox1, 2, and 4) is the largest source of O2‒• in the kidneys,11,12 but mitochondrial overproduction of O2‒• also leads to considerable oxidative stress. Oxidants (including O2‒•) are produced in the kidneys by fibroblasts, endothelial cells, vas-cular smooth muscle cells, mesangial cells, tubular cells, and po-docytes.11 Furthermore, the presence of an inflammatory process in the kidneys and/or hyperglycemia can result in the priming of neutrophils, activating the oxidative burst of these cells (i.e., ac-tivation of Nox2 system) and forming O2‒•, which leads to the formation of other ROS/RNS.13 NADPH oxidase expression is in-

creased in the glomeruli and distal tubules in experimental models of DM with nephropathy.14 Mesangial cells express p22phox and p47phox, and the activity of these subunits in a high-glucose me-dium has been implicated in the oxidative stress associated with the pathogenesis of diabetic nephropathy.15-17

Moreover, in another chronic complication from DM, a de-crease in the human body’s antioxidant defense (i.e., superoxide dismutase [SOD], catalase, and glutathione [GSH]) can occur with this condition and synergize with the oxidative damage ex-perienced by patients with DM. Therefore, improving the anti-oxidant system through a combination therapy with exogenous antioxidants may represent an important alternative for treating and/or preventing diabetic nephropathy or other DM complica-tions caused by oxidative stress.

Since 1964, when Emmerson and Howard-Flanders reported that nitroxides could provide radioprotection vis-à-vis lethal ef-fects of radiation, the recognized therapeutic potential of these compounds has been extensively studied, and currently, these studies are taking place in the medical field, where several bioac-tivities have been described.18-20 By reactions involving the trans-fer of electrons, nitroxides can be reduced to hydroxylamines or oxidized to oxoammonium cations, with an ability to react direct-ly or indirectly with ROS/RNS, thus being able to protect cells, tissue, and organs from damage induced by oxidants.18‒20,21

Among the stable nitroxide radicals, the spin label 2,2,6,6-te-tramethylpiperidine 1-oxyl (tempo) and its water-soluble ana-logue, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol) (Fig. 1), are powerful scavengers of ROS/RNS, with outstanding SOD-like activity and pleiotropic effects, which differs greatly from monofunctional antioxidants that mainly operate via a sin-gle mechanism. These compounds could have great relevance in the management of diabetic patients by controlling DM compli-cations, such as nephropathy, in which ROS/RNS contribute to the oxidative damage. Indeed, since hyperglycemia is known to induce oxidative stress through the increased production of ROS/RNS, the antioxidant action of nitroxides could lead to a decrease in these oxidants, which lowers the risk of oxidant attacks on vi-tal macromolecules and hence avoiding lipid peroxidation, pro-tein oxidation, DNA damage, and the irreversible formation of AGEs.22

Thus, the objective of this study was to evaluate, in Wistar rats with type 1 DM, the ability of the nitroxides tempol and tempo to prevent hyperglycemia, oxidative damage in the kidneys, and other complications from DM, including renal dysfunction (as in-dicated by creatinine levels), liver dysfunction (as indicated by ALT, AST, and steatosis), heart injury (direct damage and LVH), and lipid profiling (total cholesterol and fractions).

aloxano. Os animais foram em seguida avaliados quanto à glicemia, peroxidação lipídica nos rins, parâmetros bioquímicos da função renal (creatinina), função hepática (aspartato aminotransferase [AST] e alanina aminotransferase [ALT] lipídios totais e esteatose) e perfil lipídico (colesterol total e frações), e uma análise histológica dos rins, fígado, coração e olhos. Resultados: O tratamento dos ratos diabéticos com tempo ou tempol preveniu a hiperglicemia (p < 0,01). Curiosamente, apenas tempo foi capaz de prevenir a peroxidação lipídica nos rins dos ratos diabéticos (p < 0,01), enquanto que também diminuiu os níveis de creatinina (p < 0,05). O Tempol preveniu danos no fígado, rins, olhos e coração de ratos diabéticos (p < 0,01) e mostrou–se efetivo na prevenção da hipertrofia ventricular esquerda (HVE). Notavelmente, tempol e tempo impediram qualquer aumento significativo nos níveis de ALT e na quantidade de lipídios totais no fígado dos ratos diabéticos. Conclusão: Tempo e tempol apresentaram grande potencial para prevenir a hiperglicemia e suas complicações em ratos com DM tipo 1, sendo que ambos os nitróxidos preveniram o aumento de marcadores de disfunção hepática. O nitróxido tempo também impediu o dano oxidativo aos rins, enquanto que tempol atenuou consideravelmente a lesão cardíaca.

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Material and methods

Experimental proceduresAnimals

In this study, 60 healthy 6‒9-week-old male Wistar rats (Rat-tus norvegicus) weighing 250‒350 g, were used. The animals were supplied by the breeding colony of the University José do Rosário Vellano and were housed with a controlled temperature (25 ± 1°C) in a light-controlled room with a 12 h light/dark cycle. All the animals were kept in collective cages, totaling six animals per cage, and were fed a standard diet (commercial feed) and wa-ter ad libitum. The animals underwent a 10-day period of accli-matization.

Induction of type 1 diabetes mellitus (DM)Briefly, according to the methodology described by Lerco et

al,23 the animals were randomly divided into five groups, with 12 animals per group, and maintained on a fasting diet for 12 hours (with only water given ad libitum), and then the test animals (groups 2 to 5) received a single dose (150 mg/kg, intraperito-neally) of a solution of alloxan monohydrate (Sigma-Aldrich Inc., St. Louis, MO, USA). In the control group (group 1), the animals received a single dose of 0.9% saline. One and a half hours later, the supply was reintroduced. Seven days later, weighing of the animals and the determination of blood glucose levels were per-formed. Those rats in which the plasma glucose levels exceeded 250 mg/dL were considered diabetic, as shown in Fig. 2.

Experimental designThe experimental design is presented in Table 1, and its sche-

matic representation is depicted in Fig. 2.

Biological samplesAfter 60 days of treatment, the animals were maintained on a

fasting diet for 12 hours and then were anesthetized (ketamine [40 mg/kg, Bayer AS and Parke-Davis®, Berlin – Bayer, Leverkusen, Germany] and xylazine [6 mg/kg, Bayer AS and Parke-Davis®), and the blood was collected. The animals were then euthanized, and the organs were removed. The heart was removed via a thor-acotomy, in which 1.34 mM KCl (6 mL) was injected into the rats’ hearts through the left ventricle (LV), and then this organ was removed. After sagittal incision, the kidneys, eyes, and liver were also removed and fixed in 10% neutral-buffered formalin (24 hours), before histological analysis.

Preparation of tissue homogenatesThe kidney homogenate was prepared as described by Jones et

al.24 The kidneys were removed and homogenized (at 4ºC) in 0.1 M phosphate buffered saline (PBS, pH 7.2) at the rate of 5 mL/g kidney. This homogenate was centrifuged (3000 g, 10 min, 4ºC), and the supernatant was subsequently used.

Analysis of blood glucose levels, lipid profiling, renal function, and liver function

Briefly, the biochemical parameters associated with type 1 DM complications were measured using standard methods and commercial kits. The blood glucose levels and total cholesterol (TC) or fractions (Triglycerides [TG] and high-density lipopro-tein [HDL]) were determined in the serum through the endpoint colorimetric method. The serum creatinine levels (renal function) were determined by the modified Jaffe method, and the meas-urement procedure was calibrated with the standard reference material (SRM) 914a of the National Institute of Standards and Technology (NIST). To evaluate the liver function, the levels of the enzymes aspartate aminotransferase (AST) and alanine ami-notransferase (ALT) in the serum were determined by the UV-kinetic method. The total lipid concentration in the liver homoge-nates was determined by using the Bligh Dyer method, and the results were reported as total lipids (%), which represented the total lipid percentage per mL of liver homogenate.25

Evaluation of lipid peroxidation in the kidneysTo evaluate the oxidative insult to lipids, the peroxidation

products that reacted with thiobarbituric acid (TBA) were meas-ured through the thiobarbituric acid-reactive species (TBARS, oxidative stress byproducts) assay, as described by Winterbourn

Figure 1. Chemical structures of Tempo (a.) and Tempol (b.), where the un-paired electron centered on the nitroxide group is highlighted.

a. Tempo

H3C CH3

CH3

CH3

CH3

OH

H3C

H3C

H3C

N

O

b. Tempol

N

O

Figure 2. Outline of the experimental design.Subtitle: Sixty healthy Wistar rats were divided into five groups (12 animals per group). Next, using alloxan (150 mg/kg, intraperitoneally, single dose), type 1 diabetes mellitus (DM) was induced in groups 2 to 5 (first week). After that, the animals were fed and watered ad libitum, and Tempo and Tempol (30 mg nitroxide/kg/day) was given by gavage to groups 5 and 6, respectively, for two months. Next, the animals were tested for oxidative damage (kidneys), serum glucose levels, biochemical parameters, and histological analysis (eyes, liver, kidneys, and heart). In the control group, treatment with metformin (50 mg/kg/day) for 60 days in group 3 was performed.

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et al.26 The aliquots (150 µL) of the homogenate from the kidneys were mixed with 1.22 M phosphoric acid (750 μL), deionized wa-ter (1350 μL), and TBA (0.67%, 750 μL); the mixture was then incubated. Next, the incubation mixture was boiled in water for 1 hour at 95°C. After cooling in an ice bath (4°C), methanol (1800 μL), 1 M NaOH (200 μL), and the sample (1000 μL) were added to a cuvette. The concentration of TBARS was estimated from the standard curve of malonic dialdehyde (MDA; from the hydrolysis of 1,1,3,3 tetramethoxypropane). The MDA levels were quanti-fied using a Varian Cary Eclipse spectrofluorometer (λ excitation = 532 nm; λ emission = 563 nm). The results were expressed as µg MDA/mol of protein. The total protein concentration was de-termined by the Bradford method.27

Histological analysis of the organsTo evaluate morphological changes of the heart, liver, kid-

neys, and eyes, the rat hearts were dissected, and the left ventricles were fixed in 10% neutral-buffered formalin (48 hours); then, the fixed specimens were processed by the conventional paraffin-em-bedding technique.28 A series of sections of 3 µm thicknesses were taken in the same plane and deposited on the slides, stained with hematoxylin and eosin (H&E) or Masson-trichrome (sections of LV) and then were morphologically analyzed with an optical mi-croscope (Nikon, TNB-04T-PL, magnifications 40 x or 100 x). The measurement of the left ventricular amplitude followed the standard criteria using the software LGMC-image, version 1.0. All the histological analyses were performed by a single exam-iner, using the double-blind method.

Data analysisAll the graphs were constructed using BioEstat software (ver-

sion 5.0, Belém, Pará, Brazil, 2007) and, except where otherwise noted, the results are expressed as the mean ± standard deviation (SD) and are representative of three independent experiments, at least in triplicate. Analysis of variance (ANOVA), followed by Tukey’s or Bonferroni’s tests for multiple comparisons of the means, was also performed using BioEstat software. The means were considered significantly different with p values of less than 0.05 (α = 0.05). The chemical structures depicted in this study were constructed using software from ACD/Labs (Advanced Chemistry Development, Inc., version 6.0).

Results

Effects of nitroxides on body weights of the diabetic animalsThe average weights for the different experimental groups are

shown in Table 2. Tempol or tempo treatment did not influence animal weight between groups as well as within groups (after final treatment).

Effects of nitroxides on glycemia The blood analyses for the glucose levels of the animals with

the different treatments are shown in Fig. 3. The blood glucose levels of the diabetic induction group were significantly higher than the non-diabetic group, whereas both tempo and tempol treatments (30 mg/kg/day) were significantly anti-hyperglycemic in the diabetic rats compared to the untreated diabetic rats.

Table 1. Experimental groups

Group Nº initial of animals

DM induction

Nº of animalsafter induction

of DMTreatment

Nº of animalsthat completed

the study

1: Control, rats without DM 12 0.9% saline 9 ---- 9

2: Control, diabetic rats 12 Alloxan 6 ---- 6

3: Diabetic rats treated with metformin 12 Alloxan 6 Metformin 6

4: Diabetic rats treated with Tempol 12 Alloxan 7 Tempol 7

5: Diabetic rats treated with Tempo 12 Alloxan 5 Tempo 5

Table 2. Lipid profile (Serum levels of total cholesterol and fractions [triglycerides and HDL]), total lipids in liver, and weight for the experimental groups

Rats without DM Diabetic rats

TREATMENTS

— — Metformin Tempol Tempo

Cholesterol (mg/dL) 69.678 a 67.525 a 64.26 a 63.5325 a 66.92 a

Triglycerides (mg/dL) 50.5 a 68.5 a 58.5 a 67.5 a 48.6 a

HDL (mg/dL) 26 a 25.25 a 27.6 a 23.25 a 34 a

Total lipids in liver (%) 1.28 b 1.48 c 1.05 a 1.15 a 0.99 a

Weight1 (g)* 353 a,A 3393 a,A 365 a,A 303 a,A 291 a,A

Weight2 (g) 382 b,A 3113 a,A 361 b,A 315 a,A 276 a,A

*Average weight for all rats per experimental group, before1 and after2 60 days of treatment; Lowercase superscript letters: comparing within the row; Uppercase superscript letters: comparing within the column; Mean values with different letters are significantly different (p < 0.05). HDL = high-density lipoprotein; DM = type 1 diabetes mellitus

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Effects of nitroxides on oxidative damage in the kidneys as well as renal function and morphology

Interestingly, only tempo was able to prevent a significant in-crease in creatinine levels in the diabetic rats relative to the untreated diabetic rats (Fig. 4A.). As observed in Fig. 4B., a high degree of glucose-induced lipid peroxidation was observed in the diabetic rats compared with the non-diabetic rats. Notably, when compared with the untreated diabetic rats, tempo treatment (30 mg/kg/day) prevent-ed lipid peroxidation significantly in the kidneys. In contrast, treat-ment of the diabetic rats with 30 mg/kg/day of tempol did not affect

lipid peroxidation in the kidneys, confirming the findings of the lev-els of creatinine. The histological analysis of the distal and proximal tubules, Bowman’s capsules, urinary tubules, glomeruli bulbs, and Malpighian bodies were assessed (Fig.s 4C. and 4D.). Compared with the healthy rats and tempol-treated diabetic rats, the diabetic rats ex-hibited nephropathy, with increases in renal fibrosis; cortex damage;

Figure 3. Influence of the nitroxides on blood glucose levels (mg/dL).Mean values for the serum glucose levels were 149.8±41.55 mg/dL in rats without DM, whereas in groups of rats with type 1 DM, the levels were sig-nificantly (p < 0.01) higher (387.25±89.31 mg/dL. Treatment of the diabetic rats with 30 mg/kg/day Tempol or Tempo significantly decreased blood glucose levels (197.33±59.53 mg/dL and 200.4±63.03, respectively) relative to the untreated diabetic rats (p < 0.01). There was no significant difference between Tempol and Tempo treatments (p = NS). ǂ: ± standard deviation; Mean values are significantly different when p < 0.05 (α = 0.05).

Figure 4. Effects of the nitroxides on renal morphology and function and oxi-dative damage in the type 1 diabetic rats. A. Creatinine levels were significantly different between the rats without DM and the diabetic rats (p < 0.05). Diabetic rats treated with Tempo (30 mg/kg per day) had lower serum creatinine levels (p < 0.05) compared with the untreated diabetic rats. Metformin or Tempol treatment did not prevent the increase in serum creatinine levels (p = NS).

C. Representative sections of the kidneys from rats in the different experi-mental groups (H&E, magnification is 40×).

D. Representative sections of the kidneys from rats in the different experi-mental groups evaluated (H&E, magnification is 100×). ǂ: ± standard devia-tion; Mean values are significantly different when p < 0.05 (α = 0.05).

B. Lipid peroxidation was significantly higher in the kidneys of the diabetic rats compared with the rats without DM (p < 0.01). Treatment of diabetic rats with Tempo (30 mg/kg per day) significantly (p < 0.01) prevented lipid peroxidation in the kidneys compared to the untreated diabetic rats. Tempol treatment did not prevent lipid peroxidation in the kidneys of the diabetic rats compared with the untreated diabetic rats (p = NS).

500

400

300

200

100

0Healthy rats Diabetic rats

Groups

Gly

cem

ia (m

g/dl

) CH3

CH3

H3C

H3C N

O

+ Tempo

H3C CH3

CH3

OH

H3C N

O

+ Tempol

1.0

0.8

0.6

0.4

0.2

0.0Healthy rats Diabetic rats

Groups

Cre

atin

ine

leve

ls (m

g/dl

) CH3

CH3

H3C

H3C N

O

+ TempoH3C CH3

CH3

OH

H3C N

O

+ Tempol

+ Metformin

0.05

0.04

0.03

0.02

0.01

0.00Healthy rats Diabetic rats

Groups

µg M

DA

/mol

of p

rote

in

CH3

CH3

H3C

H3C N

O

+ TempoH3C CH3

CH3

OH

H3C N

O

+ Tempol

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dilation of the urinary space, the space lying within Bowman&#39;s capsule, as well as the cortical veins; glomeruli shrinkage; wrinkling of the Malpighian body in some parts; and tubular damage (cell lyses, tubular necrosis, loss of the brush border, cast formation and tubular dilatation at the corticomedullary junction).

Effects of nitroxides on ALT, AST, and liver function and morphology Both nitroxides had preventive effects on the increase in ALT

levels in diabetic rats (Fig. 5A.), whereas an increase in ALT levels was observed in the untreated diabetic rats when compared to the healthy animals. Neither tempol nor tempo changed the AST levels in diabetic rats relative to the untreated animals (data not shown). In the liver sections, the area around the port, the hepatocytes, the sinu-soids, the space around the sinusoids, the interstitial area, and the ar-eas around the central veins were evaluated (Fig. 5B.). There was a difference in the intensity of the hepatic damage. Mild to moderate

liver fibrosis (around the perisinusoidal spaces and the portal area) was observed in diabetic rats, as well as distortions of the liver ar-chitecture, with fatty liver changes (e.g., hepatocytes with fat accu-mulation, mild-moderate steatosis). The degree of fibrosis observed in the hepatic tissues was lower in the treated groups (metformin or tempol). Clear structures of the hepatic lobules and hepatocytes radially arranged around the central vein, as well as no obvious in-flammatory cell infiltration (or plasma influx) or Kupffer cell prolif-eration, appeared in the control and tempol-treated groups.

Effects of nitroxides on lipid profilingThe total cholesterol, triglycerides, and HDL did not change

significantly in the diabetic rats compared to the non-diabetic rats (Table 2). However, the percentage of total lipids increased sig-

Figure 5. Influence of the nitroxides on liver morphology and function.A. ALT levels were significantly different between the rats without DM and the diabetic rats (p < 0.01). Metformin, Tempol, or Tempo treatment signifi-cantly prevented the increase in ALT levels in the serum of the diabetic rats relative to the untreated diabetic rats.

Figure 6. Left ventricular dimensions (left ventricular wall thickness, in µm) and cardiac morphology in the evaluated groups.A. Diabetic and non-diabetic rats were significantly different regarding their left ventricular dimensions (p < 0.01). Tempol or metformin treatment significantly (p < 0.01) prevented left ventricular hypertrophy (LVH) in the diabetic rats compared to the untreated diabetic rats. ǂ: ± standard deviation; Mean values are significantly different when p < 0.05 (α = 0.05).

B. Representative sections of the liver from rats in the different experimental groups evaluated (H&E, magnification is 40×). Inflammation (and plasma cell influx) around the portal área, often seen in sinusoid cells around the central vein and sinusoids, occurring also vacuoles in some cells and cel-lular swelling (hydropic degeneration). Inflated and dilated sinusoid spaces (including parts of the sinusoids that were swollen), and cells that were less eosinophilic (diffuse basophilic focus because of the lower glycogen stores) were observed as well. ǂ: ± standard deviation; Mean values are significantly different when p < 0.05 (α = 0.05); ALT = alanine aminotransferase.

B. Magnification (100×) of the hearts from rats in the different experimental groups. Representative photomicrographs of the healthy rats (normal car-diomyocytes [1]) were compared to those from the rats presenting dispa-rate pathological changes (cardiac hypertrophy [2]). DM-induced cardiac hypertrophy was attenuated by Tempol [3] or metformin treatment [4]. Scale bars, 10 μm.

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nificantly in the liver of the diabetic rats compared to the non-diabetic rats, whereas tempo or tempol (30 mg/kg/day) treatment lowered the levels of total lipids in the diabetic rats compared with the untreated diabetic rats (Table 2), thus confirming the histologi-cal findings in the liver sections.

Influence of the nitroxide tempol on heart morphology The positive effects of tempol on the attenuation of cardiac in-

jury included the prevention of the LVH (analysis of left ventricu-lar thickness) in diabetic rats, and they are shown in Fig.s 6A and 6B. The global left ventricular thickness (Fig. 6A.) was decreased by this nitroxide, confirmed further in Fig. 6B. (normal cardiomy-ocytes, photomicrograph 3). This positive effect was also elicited by metformin treatment (Fig. 6A. and B. [photomicrograph 4]).

Effects of the nitroxide tempol on rat eyes In comparing the eye sections, the ganglion cell layer, the inner

nuclear layer, the outer nuclear layer, and the choroid were evalu-ated (Fig. 7). In the eyes, alterations were observed in the untreated diabetic rats, whereas tempol or metformin treatment relieved these types of damages in diabetic rats, which was further attested by the macroscopic observation of the eyes (data not shown).

Discussion

In this study, we undertook the testing of the nitroxides tempo and tempol—SOD mimetics and pleiotropic antioxidants— as anti-hyperglycemic agents that prevent DM complications, such as oxidative damage in the kidneys, liver dysfunction, and eye and heart injuries. The effects of these synthetic antioxidants admin-istered by gavage at a dosage of 30 mg/kg/day for 60 days were evaluated in a rat model of type 1 DM. Treatment of the diabetic rats with tempol or tempo decreased the blood glucose levels ap-proximately two-fold.

It has been reported that tempol increases glucose transport and glucose uptake by cells.29 According to Shahidi et al,2 tempol can decrease plasma glucose levels and improve lipid profiling in an experimental animal model using Wistar rats with streptozotocin-induced diabetes, in which rats were fed this nitroxide (30 mg/kg/day) for 60 days. However, in evaluating the effects of tempol on

the pancreas in an experimental acute pancreatitis model using male Wistar albino rats, Erbıs et al30 showed that this nitroxide did not lower glucose levels, despite its protective and regenerative effects on the pancreas, with the reestablishment of organ function in rats with acute necrotizing pancreatitis. In our study, nitroxides did not alter body weight. According to Zou et al,31 tempol did not normal-ize body weight or blood glucose in Akita mice, which is a well-established animal model of diabetic retinopathy.

Diabetes mellitus is markedly characterized by hyperglyce-mia and can be associated with dyslipidemia and disturbed liver and kidney functions. Interestingly, although outstanding dyslipi-demia did not occur in the rats evaluated in this study, when liver function (ALT levels) was evaluated, increased values of ALT and total lipids were observed in diabetic rats relative to the non-di-abetic rats, whereas tempo or tempol treatments prevented these increases. These data are remarkable and demonstrate the effects of the nitroxides on liver function during DM.

The NADPH oxidase-derived ROS are implicated in renal diseases and DHD.3 Increased oxidative stress in the kidneys has been recognized in different pathophysiological conditions, such as ischemia-reperfusion, CRF, hypertension, and diabetic nephropa-thy. Furthermore, it is a well-accepted idea that the high blood glu-cose-induced overproduction of ROS is present in some diseases, such as renal complications from DM.6,32 Cellular injury in renal diseases can be mediated by lipid peroxidation, which causes mem-brane damage, as well as oxidative modification of other critical intracellular targets. Antioxidants have been shown to protect the polyunsaturated fatty acids (a component of cell membranes) from oxidant attack in diabetes and to end peroxidation events.2 In this study, tempo markedly prevented lipid peroxidation in the kidneys of type 1 diabetic rats, whereas the same did not occur when diabet-ic rats were treated with tempol. The process of lipid peroxidation occurs in three steps. Unlike other antioxidants, nitroxides can in-hibit lipid peroxidation by participating in redox reactions at every step, and this bioactivity can be attributed to their property as ROS scavengers, yet these compounds present pleiotropic actions that completely mitigate the renal damage.

The effect of tempo in preventing oxidative stress-induced kidney injury was investigated further by evaluating the param-eters of renal injury, including creatinine levels and oxidative stress-induced lipid damage. A profound increase in creatinine levels occurred in diabetic rats when compared to healthy rats. Tempo not only decreased lipid peroxidation (oxidative damage in kidneys) but also prevented an increase in creatinine levels. Curi-ously, tempol did not prevent oxidative damage in the kidneys, and consequently, creatinine levels increased. These findings are in line with a recent report by Ergin et al,33 who demonstrated that tempol does not alleviate lipid peroxidation (MDA levels) in renal tissue in a rat model of renal ischemia/reperfusion by aortic clamp-ing, although the nitroxide alleviated inflammation (decreased Il-6 and iNOS) and kidney injury. Tempo is approximately 200 times more lipophilic than tempol, which may facilitate its accumula-tion in the cell membrane and explain the effect of this nitroxide in preventing lipid damage.

The effects of nitroxides on renal function have been shown in recent studies. Camacho and Israel34 reported that tempol af-fects kidneys positively by promoting an increase in antioxidant enzyme activities in an experimental model of preeclampsia, a condition in which the role of NO and its dysregulation are high-lighted. They showed that tempol improved mean arterial pres-sure, increased SOD and glutathione peroxidase (GPX) activities, and ameliorated renal function. Additionally, Ding et al,35 using a

Figure 7. Histological analysis of rat eyes.Magnification (100×) of H&E-stained sections of the eyes from rats in the different experimental groups.

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model of chronic kidney disease (CKD) induced in mice, demon-strated the effects of tempol on oxidative stress, having observed that this nitroxide inhibited NF-κß-mediated inflammation, trans-forming growth factor-ß (TGF-ß), and Smad3-induced renal fibro-sis; decreased renal markers of oxidative damage and expression of NADPH-oxidase related subunits (p47phox, p67phox, gp91phox); and prevented activation of the redox-sensitive epidermal growth factor receptor (EGFR) pathway and the mitogen-activated pro-tein kinase (MAPK) (cRaf/MEK/ERK) signaling pathway, ac-cordingly attenuating renal injury in these mice.

The most serious renal lesions have been described as occur-ring from 3 to 30 weeks after the onset of untreated DM, a time frame covered in this study; considerable morpho-pathological alterations and changes in biomarkers of renal function have been observed at four weeks.36 As another example, Banday et al,37 in evaluating the influence of tempol on obese Zucker rats, carried out tests after 15 days of treatments with this nitroxide. Within this time interval, they found that tempol caused a decrease in oxidative stress, an improved sensitivity to insulin, and a restored function of the D1 and G-protein coupling receptors.

Complications of DM include coronary heart disease, which is currently strongly linked to LVH.4 As demonstrated in this study, tempol markedly prevented LVH in diabetic rats, further corrobo-rating a similar effect presented by this nitroxide on LVH using an experimental model of LDL receptor gene knockout (LDLr-/-) mice with a high-fat diet.28 We also demonstrated that metformin (used for treating type 2 DM) decreased LVH and yet had no anti-hyperglycemic effect (data not shown). Previous reports relate the positive effects of metformin on preventing types 1 and 2 DM-associated complications, including decreased levels of AGES/advanced oxidation protein products (AOPPs), oxidative damage, and biochemical parameters, notwithstanding its inability to elicit an anti-hyperglycemic effect on type 1 DM.39

Chronic alterations of the eye in uncontrolled type 1 DM in-clude retinopathy and blindness, among others. In our study, tem-pol decreased such alterations in diabetic rats, as also reported by Zarling et al 40;Thaler et al, 41 who evidenced protective effects of tempol on alterations of the eye and proposed the pharmaceutical development of nitroxides that show a potential for treating age-related degeneration and disease.

Regarding the dosages of nitroxides in the present study, these compounds present very low toxicity in vitro and in vivo, having no toxic effects at a dosages of 30 mg/kg/day20; thus, their safe use can be justified. Moreover, in this study, no death related to the treat-ments with the nitroxides occurred (i.e., all of the tempo or tempol-treated animals survived at the 68th day of experiments). Concern-ing the metabolism, the most common site where the reduction of nitroxides occurs is in the liver’s mitochondrial respiratory chain and the microsomal electron transport system, where glucose ad-ministration and mitochondrial density (which is related to ROS production) enhance the activity of these systems by increasing the reduction of nitroxides, which could explain the difference in the results between the evaluated nitroxides and could take into account that the renal use of glucose is independent of insulin.20,42,43

Our results indicate that, for an experimental alloxan-induced type 1 DM, tempo and tempol present anti-hyperglycemic effects, although only tempo ameliorates renal disfunction by reducing oxidative damage and improves renal filtration with a remarkable reduction in creatinine levels, thus shedding light onto new adju-vant therapies for DM. Tempol ameliorated the heart morphologi-cal complications (effect on LVH) resulting from DM. The pleio-tropic effects of these nitroxides converge on the final beneficial

role observed: the primary effects of maintaining blood glucose homeostasis and attenuating lipid peroxidation, as evidenced in this study, as well as the effects reported in earlier studies, such as the control of macrovascular and microvascular changes, the con-trol of key metabolic pathways in the development of DM compli-cations, and the local protective effects on renal circulation as well as for preventing oxidative stress-induced organ injury [especially acting by diminishing NADPH oxidase complex-generated ROS, through mechanisms up-stream of this complex and/or by directly scavenging ROS, according previous studies by our group and others18,20,44,45)]. Therefore, the beneficial activities of these nitrox-ides may offer new options for pharmacological interventions in the treatment of diseases with excessive ROS/RNS production, such as what occurs during DM complications. The roles of these nitroxides in humans should be investigated further.

Highlights:(1) Nitroxides improve biochemical parameters in rats with

diabetes mellitus (DM)(2) Tempo prevents DM-associated oxidative stress-induced

renal injury(3) Tempol abrogates DM-related left ventricular hypertrophy(4) Tempol prevents eye, liver and renal injuries

Acknowledgments/Agradecimentos: We would like to thank Prof. Ana Claudia Frota Machado de Melo Lopes for a kind help in the analysis of the histological slides.

Responsabilidades Éticas

Conflitos de Interesse: Conflitos de interesse na realização do presente trabalho. Fontes de Financiamento: Não existiram fontes externas de fi-nanciamento para a realização deste artigo.Proteção de Pessoas e Animais: Os autores declaram que os procedimentos seguidos estavam de acordo com os regulamentos es-tabelecidos pelos responsáveis da Comissão de Investigação e Ética. Proveniência e Revisão por Pares: Não comissionado; revisão externa por pares.

Ethical Disclosures

Conflicts of Interest: The authors report no conflict of interest. Funding Sources: No subsidies or grants contributed to this work. Protection of Human and Animal Subjects: The authors declare that the procedures followed were in accordance with the regula-tions of the relevant research ethics committee. Provenance and Peer Review: Not commissioned; externally peer reviewed. References / Referências

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